Mitogen activated protein kinase (MAPK) cascades are comprised of three sequentially acting protein kinases. Related cascades are reiterated in the cell to mediate distinct responses to a host of extracellular stimuli. Despite the potential this situation presents for extensive cross talk, it is generally observed that the MAPKs of a given pathway are activated only by the appropriate stimuli. This specificity appears to be maintained because the enzymes of a given cascade are associated with each other in stable complexes. The ramifications that this modular organization has on signal amplification, regulation and integration is central to understanding events that control normal proliferation, development and inflammatory responses. While a modular organization of vertebrate pathways is becoming apparent, analogous pathways in yeast are the paradigms for the architecture and dynamics of these cascades. We will exploit what is known about the yeast mating stress and cell- integrity cascades to learn what consequences a modular organization has on the mechanics of signal transmission and on inter-pathway communication. Our aims are to: [1] Define the regulation and function of scaffold associations in signaling. Mutants of the MAPK kinase Ste7 that are defective for binding to the scaffold-Ste5 will be isolated to identity the binding region and learn how phosphorylation regulates the Ste7-Ste5 association. The mutants also will be used as tools to learn what role the Ste7-Ste5 association has on signaling in vivo and on phosphotransfer reactions in vitro. [2] IdentifY protein(s) controlling transitions between on and off states of signaling assemblies. Two hybrid screens and a biochemical approach will be used to isolate regulators that facilitate a transition from a non-productive to productive interaction of Ste7 with other components of the mating pathway module. [3] Investigate whether distribution of shared components regulates pathway activities. Binding defective mutants of the MAPK kinase kinase Ste11 that discriminate between binding to Ste5 in the mating module and the MAPK kinase Pbs2 in the stress module will be isolated and used to learn if its interaction with one module limits signaling in the other. Pbs2-Ste11 co-localization will be examined in vivo during signalling using confocal fluorescence microscopy. {4} Define the mechanism by which one stimulus activates two MAPK pathways. Pheromone activates the mating MAPK cascade whose output then stimulates the cell-integrity MAPK cascade. Genetic methods will be used to identity the "second messengers" coordinating the of activities of these two pathways.